Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)
Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)
Hussam ZaherID 0 2
Robert W. Murphy 1 2
Juan Camilo Arredondo 0 2
Roberta Graboski 0 2
Paulo Roberto Machado-Filho 0 2
Kristin Mahlow 2
Giovanna G. Montingelli 0 2
Ana Bottallo Quadros 0 2
Nikolai L. Orlov 2
Mark Wilkinson 2
Ya-Ping Zhang 2
Felipe G. Grazziotin 2
0 Museu de Zoologia, Universidade de Sa?o Paulo , Sa?o Paulo, Sa?o Paulo, Brazil, 2 CR2P - Centre de Recherche en Pale ? ontologie - Mus e ?um national d'Histoire naturelle - Sorbonne Universite ? , Paris , France
1 Centre for Biodiversity, Royal Ontario Museum , Toronto, Ontario , Canada , 4 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology , Kunming, China, 5 Laborato ?rio de Herpetologia, Museu Paraense Em ??lio Goeldi, Bele ? m, Par a ?, Brazil, 6 Museum fu ? r Naturkunde , Leibniz Institute for Evolution and Biodiversity Science , Berlin, Germany , 7 Zoological Institute, Russian Academy of Sciences , Saint Petersburg , Russia , 8 Department of Life Sciences , The Natural History Museum, London , United Kingdom , 9 Laboratory for Conservation and Utilization of Bio-resources, Yunnan University , Kunming, China, 10 Laborat o ?rio de Colec ?o?es Zool o ?gicas , Instituto Butantan , Sa?o Paulo, Sa?o Paulo , Brazil
2 Editor: Ulrich Joger , State Museum of Natural History , GERMANY
Caenophidian snakes include the file snake genus Acrochordus and advanced colubroidean snakes that radiated mainly during the Neogene. Although caenophidian snakes are a wellsupported clade, their inferred affinities, based either on molecular or morphological data, remain poorly known or controversial. Here, we provide an expanded molecular phylogenetic analysis of Caenophidia and use three non-parametric measures of support-Shimodaira-Hasegawa-Like test (SHL), Felsentein (FBP) and transfer (TBE) bootstrap measuresto evaluate the robustness of each clade in the molecular tree. That very different alternative support values are common suggests that results based on only one support value should be viewed with caution. Using a scheme to combine support values, we find 20.9% of the 1265 clades comprising the inferred caenophidian tree are unambiguously supported by both SHL and FBP values, while almost 37% are unsupported or ambiguously supported, revealing the substantial extent of phylogenetic problems within Caenophidia. Combined FBP/TBE support values show similar results, while SHL/TBE result in slightly higher combined values. We consider key morphological attributes of colubroidean cranial, vertebral and hemipenial anatomy and provide additional morphological evidence supporting the clades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise the relevant caenophidian fossil record and provide a time-calibrated tree derived from our molecular data to discuss the main cladogenetic events that resulted in present-day patterns of
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
caenophidian diversification. Our results suggest that all extant families of Colubroidea and
Elapoidea composing the present-day endoglyptodont fauna originated rapidly within the
was provided by Fundac??o de Amparo ? Pesquisa
do Estado de S?o Paulo (BIOTA/FAPESP grants
2002/13602-4 and 2011/50206-9 to HZ and 2016/
50127-5). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
early Oligocene?between approximately 33 and 28 Mya?following the major terrestrial
faunal turnover known as the ?Grande Coupure? and associated with the overall climate shift at
the Eocene-Oligocene boundary. Our results further suggest that the caenophidian radiation
originated within the Caenozoic, with the divergence between Colubroides and
Acrochordidae occurring in the early Eocene, at ~ 56 Mya.
Determining the phylogenetic affinities within snakes was viewed by many herpetologists in
the past as an insurmountable challenge. Underwood  expressed his profound frustration
with a simple sentence: "I have found snake systematics to be a hard test to intellectual
honesty?. Although the phylogenetic affinities of snakes were indeed difficult to determine on
morphological grounds, monophyly of some higher-level taxa represent a long-standing
consensus. This is the case for the clade Caenophidia, a group of advanced alethinophidian snakes
recognized formally by Hoffstetter [
] to accommodate the families Colubridae, Dipsadidae,
Hydrophiidae, Elapidae, and Viperidae. Hoffstetter?s Caenophidia was characterized by the
absence of a coronoid bone and included the colubrid subfamily Acrochordinae, already
known to share several additional derived morphological traits with the remaining
caenophidian families [
]. The same group of ?advanced alethinophidian snakes? was also recognized
by Romer [
], who preferred to accommodate them in a newly erected superfamily
Colubroidea, equating the latter with Hoffstetter?s concept of Caenophidia. ?Acrochordoids? and
?colubroids? were only later recognized as two distinct superfamilies within Caenophidia after
] argued convincingly that acrochordids were the sister-group of the
remaining caenophidians based on a number of synapomorphies derived from the vomeronasal
capsule, musculature, hyoid and costal cartilages [
Molecular phylogenies ultimately provided strong support for the monophyly of
Caenophidia, and further corroborated more controversial morphological hypotheses, such as the
polyphyly of solenoglyphous [
] and proteroglyphous snakes [
]. On the other hand,
analyses of molecular evidence also obtained conflicting results for the positions of acrochordids
and xenodermids at the base of the Caenophidian tree and highlighted the need of
substantial taxonomic changes in order to obtain monophyletic familial level taxa [
despite notable advances, many questions regarding the higher-level phylogeny and
taxonomy of Caenophidia remain unanswered, and a period of taxonomic instability has seen a
number of different, and sometimes contradictory, classification schemes, with none of them
being entirely satisfactory (S1 Table).
Three large-scale molecular phylogenies of snakes were published recently [
However, despite their impressive taxon sampling, substantial overlap in data and similar analytical
strategies, these studies have produced a surprisingly large number of differences in inferred
relationships at the familial and generic levels (Figs 1?3). Pyron et al. [
] and Figueroa et al.
] based a number of taxonomic actions exclusively on their molecular phylogenetic analyses
with no attempt to reconcile these with the available morphological and paleontological
evidence. This is understandable given that one of the main advantages of molecular over
morphological phylogenetics is the wider coverage of species that the technique allows within a
relatively short amount of time.
Large-sample comparative morphological studies are often difficult to achieve due to the
need for destructive investigative procedures on limited museum specimens and by the lack of
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Fig 1. Higher-level caenophidian phylogenies. Comparison between Maximum-likelihood phylogenetic estimates from (A) the
present study and (B) Figueroa et al. [
]. Tips represent commonly recognized families, subfamilies and rogue taxa. Names in
red correspond to taxa with distinct phylogenetic positions in the topologies compared. Numbers on each branch and within
expanded tips correspond to our and previously reported support values: (A) FBP (left) and SHL (right); (B) FBP. Branches
without numbers have support <70%.
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Fig 2. Higher-level caenophidian phylogenies. Comparison between Maximum-likelihood phylogenetic estimates from (A)
the present study and (B) Pyron et al. [
]. Tips represent commonly recognized families, subfamilies and rogue taxa. Names
in red correspond to taxa with distinct phylogenetic positions in the topologies compared. Numbers on each branch and
within expanded tips correspond to our and previously reported support values: (A) FBP (left) and SHL (right); (B) SHL.
Branches without numbers have support <70%.
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Fig 3. Higher-level caenophidian phylogenies. Comparison between Maximum-likelihood phylogenetic estimates from (A)
the present study and (B) Zheng and Wiens [
]. Tips represent commonly recognized families, subfamilies and rogue taxa.
Names in red correspond to taxa with distinct phylogenetic positions in the topologies compared. Numbers on each branch
and within expanded tips correspond to our and previously reported support values: (A) FBP (left) and SHL (right); (B) FBP.
Branches without numbers have support <70%.
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comprehensive taxonomic coverage in skeletal collections all over the world. Morphological
information on caenophidian snake anatomy is still very limited compared to the diversity
within the group, with only a few detailed analyses of larger clades being available in the
literature and mainly focused on the cranial and hemipenial complexes [
]. A notable exception
is the monographic study of Cundall and Irish  of the skull of snakes, which provides the
first comprehensive large-scale comparative analysis of caenophidian cranial anatomy.
The paleontological record for caenophidian snakes is largely biased towards disarticulated
postcranial (vertebral) materials [
]. Although caenophidian vertebral elements are
frequently found in Caenozoic vertebrate-bearing deposits, their identification at the generic and
familial levels are often difficult to ascertain, mostly because of our limited knowledge of
vertebral morphology and its variation within caenophidian families. However, although limited,
our present knowledge on the cranial, vertebral, and hemipenial anatomy of the group still
constitutes an important body of evidence that can be evaluated within an explicit molecular
phylogenetic framework, helping highlight major events in the origin and diversification of
caenophidian snakes. This approach can help circumvent conflicts between multiple
alternative molecular hypotheses of relationships [
] that seem to be correlated with poorly
sampled groups or short internal branches combined with terminal taxa with long branches
resulting from the accumulation of molecular autapomorphies .
Here, we provide an expanded molecular phylogenetic tree of Caenophidia, highlighting
strongly and weakly supported hypotheses of relationships that need further investigation. We
evaluate alternatively three non-parametric support values?Shimodaira-Hasegawa-Like test
(SHL), Felsentein bootstrap proportions (FBP), and transfer bootstrap expectation metrics
(TBE)?and combine two of these (FBP and SHL) to give a seven-category classification of the
robustness of clades in the molecular tree (S2 Table). Contradictory support values are
frequently encountered, suggesting that results based on only one of these three support values,
should be viewed with caution. We use our molecular tree as a backbone phylogeny to review
some key morphological characters of caenophidian snakes in an attempt to reconcile both
morphological and molecular bodies of evidence at the familial and suparfamilial levels of the
tree. We focus on two main anatomical complexes in the skull of caenophidian snakes?the
optic nerve foramen/fenestra and the naso-frontal joint?known to be phylogenetically
informative at higher levels [
]. We also revise anatomical evidence from the vertebrae and
hemipenes of representatives of all known extant colubroidean families. Finally, we combine
information from the known fossil record and a time-calibrated tree derived from our
molecular data to discuss the main cladogenetic events that resulted in present-day patterns of
Materials and methods
Lawson et al. [
], Zaher et al. [
], and Pyron et al. [
] provided a listing of extant genera
considered valid under their family-group names, while Uetz et al. [
] and Wallach et al. 
went further and compiled complete listings of all extant species. In addition to known extant
taxa, Wallach et al.  also provided a listing of extinct genera and species. Uetz et al. [
species list represents a compilation of valid names that follows, in most respects, the latest
taxonomic opinions, and thus can be highly unstable. On the other hand, Wallach et al.?s 
work includes a large number of changes and corrections based on their own critical
taxonomic opinion. In that sense, the latter work is a valuable source of original information.
However, Uetz et al. [
] taxonomic list seems to integrate more accurately the massive
contribution of the Herpetological community in recent years and we use that as a framework to
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describe our results with respect to valid genera and species. However, we consider both
taxonomic schemes at the family level to be problematic in several respects and follow instead the
supra-familial and familial taxonomic scheme proposed by Zaher et al. [
], expanded here
to include recently erected or recognized Pseudoxyrhophiinae, Grayiinae, Prosymnidae, and
]. We also followed Savage  in the use of the family names Pareidae
and Xenodermidae instead of Pareatidae and Xenodermatidae. Recently, Weinell and Brown
 resolved the phylogenetic affinities of the genera Cyclocorus and Oxyrhabdium, which
along with Hologerrhum and Myersophis, were retrieved in their analysis as a well-supported
elapoid clade of endemic Philippine snakes. Weinell and Brown  accommodated these
four genera in a new subfamily, Cyclocorinae, further referred herein as a family,
Cyclocoridae. As a result, we considered the following 19 families here: Xenodermidae, Pareidae,
Viperidae, Homalopsidae, Elapidae, Psammophiidae, Atractaspididae, Pseudoxyrhophiidae,
Lamprophiidae, Cyclocoridae, Prosymnidae, Pseudaspididae, Sibynophiidae, Calamariidae,
Grayiidae, Colubridae, Pseudoxenodontidae, Dipsadidae, and Natricidae.
The potential taxonomic instability generated by unstable species or species groups
representing rogue taxa  in molecular analyses is also of concern here. Recently, many of these
taxa were given new generic or subgeneric names by R. Hoser who?s approach is considered
unethical and potentially harmful for taxonomic stability, resulting in a request by a large
consorsium of herpetologists, that the International Commission of Zoological Nomenclature
(ICZN) invalidate these new names , a petition we strongly support. We refrain from
using these names until a definitive decision on the validity of such names is reached by the
Taxon and gene sampling
We assembled a data matrix comprising 1278 (15 outgroup and 1263 ingroup) terminal
taxa representing all caenophidian families (see S3 Table for number of genes and accession
numbers; see also S4 and S5 Tables for more details on taxon sampling). We obtained 5063
sequences from GenBank and generated 1384 new sequences for up to 15 genes, including
six mitochondrial (12S, 16S, cox1, cytb, nd2, nd4) and nine nuclear (amel, bdnf, c-mos, jun,
hoxa13, nt3, r35, rag1, rag2) loci, for a total of 6447 sequences for 15 genes (S3 Table).
More than 640,000 nucleotide sequences for snakes are currently deposited in GenBank,
representing an unparalleled resource for studies of the genetic diversity of the group.
However, the quality and reliability of these data are a concern because of misidentification,
mislabelling, and sequence contamination which seem to be the principal sources of error present
in public databases . In our search for sequences of Caenophidian snakes deposited in
GenBank, we found 38 problematic sequences that were not included in the present analysis.
All questionable sequences are reported in the supporting information (S6 Table) with
succinct descriptions of the possible problems affecting each rejected sequence.
New sequences generated in this study represented 21% of our whole matrix (S3 Table),
with more than 50% and up to 80% of new sequences added to previously known sequences
for genes amel, bdnf, nt3, jun, and hoxa13, while no sequences were generated for cox1, nd2,
nd4, r35, and rag2 (S3 Table). Newly added sequences are illustrated with colored diamonds
on each tip of terminals, representing the percentage of data generated in this study (white,
0%; light gray, between 1% and 50%; dark gray, between 50% and 99%; black, 100%). We did
not sample all terminals for all genes, and the percentage of missing terminals varied from
16% for cytb to 93% for amel (S3 Table). The total number of species for families and
subfamilies recognized in Uetz et al. [
] are summarized as supporting information (S5 Table). Tissue
samples were obtained from museum collections.
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According to Uetz et al.?s [
] generic and specific taxonomic listing, our complete taxon
sampling of colubroids includes 78% and 42% of all recognized genera and species,
respectively, totaling 344 genera and 1263 species (S4 Table). From this total of 1263 species, 20
species are recognized here but not listed by Uetz et al. [
] (S7 Table). According to these slightly
revised numbers, summarized in S4 Table, we have the following generic and specific
representations in percentage, respectively: Colubridae (77% and 37%), Dipsadidae (81% and 31%),
Elapidae (85% and 54%), Homalopsidae (57% and 47%), Lamprophiidae (83% and 42%),
Natricidae (68% and 39%), Pareidae (100% and 60%), Pseudoxenodontidae (100% and 40%),
Viperidae (97% and 73%), Xenodermidae (67% and 28%).
Outgroup sampling included representatives of the following families (number of terminals
in parenthesis): Acrochordidae (3), Aniliidae (1), Boidae (2), Bolyeriidae (1), Calabariidae (1),
Cylindrophiidae (1), Erycidae (1), Loxocemidae (1), Pythonidae (1), Uropeltidae (1) and
Xenopeltidae (1). Trees were rooted with the typhlopid Indotyphlops braminus.
DNA was extracted from scales, shed skin, liver or muscle tissues using the phenol:chloroform
method following specific protocols for each tissue [40,41]. PCRs were performed using
standard protocols  for 11 genes, including four mitochondrial (12S, 16S, cox1, cytb) and seven
nuclear (amel, bdnf, c-mos, jun, hoxa13, nt3, rag1). The sequences for each pair of primers and
their respective references are provided as supporting information (S8 Table). PCRs were
purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ).
Sequences were generated in Brazil at the Laborato?rio de Biologia Geno?mica e Molecular,
Pontif??cia Universidade Cato?lica do Rio Grande do Sul (Porto Alegre, Rio Grande do Sul) using
the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated
sequencer (GE Healthcare); and in China at Laboratory for Conservation and Utilization of
Bio-resources, Yunnan University (Kunming, Yunnan) using BigDye Terminator cycle
sequencing kit in an ABI 3700 sequencer (Applied Biosystems, Foster City, CA). Both strands
were sequenced for all fragments and sequences were edited and assembled using Geneious
5.5 (http://www.geneious.com) .
Sequences were aligned using MAFFT version 6  applying the E-INS-i algorithm for
rRNAs (12S and 16S) and the FFT-NS-i algorithm for protein coding sequences. The scoring
matrix for nucleotide sequences was set to 200PAM/k = 2 and gap opening penalty was set to
1,53. Because sequences from GenBank present significant differences in size, we aligned them
using a specific procedure that accounts for blocks of overlapping sequences to avoid
alignment errors in both extremities of the aligned sequences. Extremities were then realigned
using the same algorithm previously applied for each separate gene.
Although our matrix retains high levels of missing data (average of 77.9%), sequences data
per taxon range from 286 to 12659 bp with an average of 3299 bp. Similarly, highly incomplete
taxa have been argued to be of minor concern in large-scale analyses that include many
informative characters [
] and, as elsewhere [
], our most highly incomplete taxa were
consistently placed in phylogenetic positions that are similar to previous works.
We used PartitionFinder v1.1.1 [
] in order to select a partition scheme and evolutionary
models based on AICc. We used the program RAxML version 7.2.8  to perform a
phylogenetic analysis employing Maximum Likelihood (ML) as the optimality criterion. We ran 1000
pseudoreplications of non-parametric bootstrap and we calculated FBP [
] using the rapid
bootstrap algorithm implemented in RAxML (-f a). This also conducts a search for the ML
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tree using each 5th bootstrap tree as a starting point for the rapid hill-climbing search (totaling
200 starting trees). Based on the 1000 trees derived from the pseudoreplications we calculated
the TBE [
] using RAxML-NG [
]. We also calculated branch support using SHL [
implemented in RAxML (option?f E) for each branch of the tree.
Comparing measures of clade support
We compared SHL, FBP, and TBE for clades in our molecular tree and used them in
combination to evaluate the robustness of specific clades [
]. We choose to use only the joint
values of SHL and FBP to comment the results and base our discussion because they produced
more conservative values than TBE (see results on comparisons of support metrics below for
more details). We classified the robustness of each clade in seven categories based on the
combined clade supports given by the SHL/FBP pair of support measures. These categories are
graphically illustrated as supporting information (S2 Table) and summarized on the upper left
corner of figures in the text, and are described as follows: 1) unambiguously supported, when
both support methods recover values of 100%; 2) robustly supported, when clade support is
not unambiguous, but both methods recover values 90%, or 80% in one method and
100% in the other; 3) strongly supported, when clade support does not reach percentages
equal to previous categories 1 and 2, but both methods recover values 80%, or values 70%
in one method and 90% in the other; 4) moderately supported, when clade support does
not reach percentages equal to previous categories 1, 2, and 3, but both methods recover
values 70%; 5) ambiguously supported, when clade support presents highly discrepant
values, with < 70% in one method and 80% in the other method; 6) poorly supported, when
clade support presents values < 70% in one method and between 70% and 80% in the other
method; 7) unsupported, when clade support presents values < 70% for both methods (S2
Although recognizing the subjectivity and arbitrariness of the described categories, we
apply this approach in order to clearly state our reasoning and to facilitate the description of
our general level of confidence in each clade retrieved by our phylogenetic analysis. Based on
our seven categories, we suggest two main groups of combined support values: a first one with
unquestionable or confident combined support values (categories 1, 2, 3, and 4), and a second
one with contradictory or unsatisfactory support values (categories 5, 6, and 7).
We highlight clades with contradictory (ambiguous) FBP/SHL support values because we
consider them to be potentially erroneous and thus problematic when used in taxonomy or as
presumptions for studies applying phylogenetic comparative methods (traits evolution),
estimations of diversification rates (speciation/extinction rates) approaches of historical
biogeography (discovery and event-based biogeography) and other methods requiring estimates of
phylogeny. These unsupported clades should be treated with caution and either the
uncertainty taken into account or their use eschewed altogether.
We revised key morphological characters from the skull, hemipenis and vertebrae in
representatives of most extant caenophidian families. Regarding the skull, we focused in two main
anatomical complexes?the optic nerve foramen/fenestra and the naso-frontal joint?known to be
phylogenetically informative in caenophidian snakes [
]. In that sense, we do not intend to
provide here a thorough revision of colubroidean anatomy, and prefer to refer to Underwood
, McDowell [
], Zaher [
], and Cundall and Irish [
] for a more complete review of the
pertinent literature related to these morphological complexes. However, we provide figures
of the relevant views of the skulls (S1 Appendix), vertebrae (S2 Appendix), and hemipenes
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(S3 Appendix) from representatives of most caenophidian groups in order to illustrate the
character states discussed herein. Specimens examined are listed in their respective supporting
information files (S1?S3 Appendices). Institutional acronyms are as follow: AMNH, American
Museum of Natural History, New York; BMNH, The Natural History Museum, London;
FMNH, Field Museum of Natural History, Chicago; HUJR, Museum of Zoology, Hebrew
University, Jerusalem; IBSP, Instituto Butantan, S?o Paulo; KU, Museum of Natural History,
University of Kansas, Lawrence; LSUMZ, Louisiana State University, Baton Rouge; MNHN,
Muse?um national d?Histoire naturelle de Paris; MZUSP, Museu de Zoologia, Universidade de
S?o Paulo; ROM, Royal Ontario Museum, Toronto; UMMZ, Museum of Zoology, University
of Michigan, Ann Arbor; USNM, National Museum of Natural History, Washington; ZMB,
Museum fu?r Naturkunde, Berlin.
Divergence time estimates
We generated a time-calibrated tree for our complete molecular data set that provided a
framework for the interpretation of paleontological, biogeographic, and cladogenetic patterns of
The large size of our molecular matrix (1278 terminals and 15 genes) precluded the use
of commonly available parametric uncorrelated relaxed clock methods, as implemented in
] and PAML . Instead, we used an autocorrelated relaxed clock method based
on a penalized likelihood implemented in the program treePL [54,55]. Divergence times were
calculated in treePL by applying a smoothing parameter that defined the penalty for shifting
the evolutionary rates among branches. This semiparametric method has been very effective
for other data sets with large numbers of taxa [56,57].
To determine the smoothing parameter, we iterated 20 times a cross-validation procedure
based on the RSRCV method (random subsample and replicate cross-validation)  with
lambda values ranging from 0.01 to 100,000 (select lambda = 10) and the thorough option to
ensure that the run iterates until convergence. We used the multicore option to distribute the
cross-validation analyses on 64 processors of a Linux server.
Since treePL only implements uniform prior distributions for node calibration points
[54,55], the option of setting an open uniform distribution (by not defining a hard lower
bound) can have two main undesirable effects: 1) estimating unrealistic older divergence
times; and 2) providing a much larger space for parameter sorting, and thus decreasing the
level of convergence of the estimation. In order to avoid these problems, we set maximum ages
based on a phylogenetic approach, which takes into consideration the age of relative
cladogenetic events. We made the assumption that the maximum age of a specific clade cannot be
older than the minimum age of its more inclusive clade. For the purpose of our analysis, two
lower bound dates were used to set uniform prior maximum ages: 93.9 Mya. (split between
Alethinophidia and Scolecophidia) as the maximum age for our calibration of
non-colubroidean nodes, and 54 Mya. (split between Colubriformes and Xenodermidae) as the maximum
age for colubroidean nodes.
Calibration points, fossils and constraint dates chosen for our divergence time analysis
were as follow:
1. Alethinophidia stem clade?Haasiophis terrasanctus Tchernov, Rieppel, Zaher, Polcyn &
Jacobs, 2000 was set as the Most Recent Common Ancestor (MRCA) of Anilius scytale
and Indotyphlops braminus. The holotype corresponds to a complete, articulated
specimen recovered from the Ein Yabrud quarries, near Ramallah, West Bank Palestinian
Territories (Hebrew University of Jerusalem Paleontological Collections, HJU-PAL EJ 695).
Hsiang et al.  combined molecular and morphological data in an unconstrained
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analysis analysis and reported that Haasiophis terrasanctus was a stem-Alethinophidia
instead of a crown-Alethinophidia and we adopted their conclusion. New interpretations
of several characters have supported this view . Because Hsiang et al.?s  analysis
incorporates the known phylogenetic uncertainties related to the position of this fossil, we
prefer to use Haasiophis as a stem-alethinophidian instead of a crown-taxa as traditionally
used. The unclear position of the Ein Yabrud quarries within the Cenomanian may fall
either in the Early Cenomanian Bet-Meir Formation  or the Late Cenomanian
Amminadav Formation . We followed Head  in using an age range that reflects the
uncertainty of the position of Ein Yabrud, by assuming the minimum age set for the
Cenomanian . Thus this root-node was set as a hard bound , and constrained to 93.9
2. Boinae stem clade?Titanoboa cerrejonensis Head, Bloch, Hastings, Bourque, Cadena,
Herrera, Polly & Jaramillo, 2009 was set as the MRCA of Boa constrictor and Eryx
colubrinus. The holotype corresponds to one single precloacal vertebra (UF/IGM 1), and referred
material includes 185 additional precloacal vertebrae and associated ribs representing 28
individuals from the Cerrejo?n Coal Mine, Rancheria River Valley, Guajira Peninsula,
Colombia . Morphology of the paracotylar foramina and a convex anterior zygosphene
margin suggested that Titanoboa cerrejonensis belongs to the Boinae [61,63,64]. Head?s 
preliminary analysis of undescribed cranial elements placed it in the stem lineage of the
boine radiation . Titanoboa cerrejonensis was recovered from sediments located within
the Palynological zone Cu-02 of the Cerrejo?n Formation, dated as Middle to Late Paleocene
[66,67]. Thus, the minimum age was constrained to 58 Mya and maximum age constrained
to 93.9 Mya.
3. Colubriformes stem clade?Procerophis sahnii Rage, Folie, Rana, Singh, Rose & Smith,
2008 was set as the MRCA of Asthenodipsas vertebralis and Achalinus rufescens. The
holotype consists of one posterior precloacal vertebra ("Rana Collection" from Vastan, VAS
1014), and referred material includes five precloacal vertebrae and two caudal vertebrae
from the Vastan Lignite mine, Gujarat, India . The lightly built and elongate shape of
the precloacal vertebrae, presence of tapering prezygapophyseal processes, and blade-like
uniformly thin neural spine that reaches the roof of the zygosphene refer P. sahnii to the
clade Colubriformes. The combination of these characteristics excludes Procerophis from
an association with the families Acrochordidae, Russellophiidae, Anomalophiidae, and
Xenodermidae (S2 Appendix). The differentiated para- and diapophysial articular facets
further distinguishes Procerophis from russellophiids and anomalophiids. The presence of a
plesiomorphic prezygapophyseal morphology, with articular facets predominantly
anteriorly angled, supports a basal position within Colubroides [
]. The rich squamate fauna
from the Vastan Mine of the Cambay Formation was recovered from thin continental
lenses of dark claystone and underlying marine shell beds, indicative of a near-shore
environment deposited about 1 m above one of the two major Lignite layers (Lignite 2) present
in the mine [
]. The squamate layer is situated approximately 14 m below the occurrence
of the age-diagnostic foraminiferan Nummulites burdigalensis burdigalensis [
indicative of shallow benthic zone SBZ 10 of Middle Ypresian age, which defines a minimum age
of ~53 Mya for the deposit [
]. However, we here follow  in constraining the age of
the vertebrate bearing bed of Vastan mine to an early-middle Ypresian (~54 Mya). The
occurrence in the section of dinoflagellate cysts of early Ypresian age (~54?55 Mya) [
and Strontium isotope age estimates for the deposits based on 87Sr/86Sr values clustering at
an age of 54 Mya  support this slightly older age. The minimum age was constrained to
54 Mya and maximum age constrained to 93.9 Mya.
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4. Viperidae stem clade?Vipera cf. V. antiqua  was set as the MRCA of Azemiops feae
and Causus lichtensteinii. A moderately well-preserved cervical vertebra (Staatliches
Museum fu?r Naturkunde, Stuttgart, SMNS uncatalogued) from Weisenau, Germany. The
presence of a long and straight, slightly posteroventrally directed hypapophysis and a large
condyle with a ventral portion lying on the posterior margin of the hypapophysis refers
unambiguously the cervical vertebra from Weisenau to the family Viperidae. However, its
assignment to the "Vipera aspis complex" by Szyndlar and Rage  is questionable since
characters that are known to be diagnostic of the "Vipera aspis complex", such as an
elongate centrum and short neural spines, are not marked in the cervical region. Additionally,
an elongate centrum and short neural spines have been reported in distantly related genus
Causus  and more closely related Daboia mauritanica . Furthermore, the traditional
subdivision of the "Vipera aspis complex", as originally proposed by Saint Girons  and
detailed by Nilson and Andre?n  and Herrmann and Joger , corresponds to a
paraphyletic arrangement of species as evidenced in our analysis (S1 Fig).
The vertebra from Weisenau, as well as those reported from Saint-Gerand-le-Puy ,
Hessler , and Amo?neburg , are all from the earliest Miocene of Europe (European
Land Mammal Age Neogene units MN 1 and MN 2), with the former likely being the
earliest record for the family (MN 1). Although the precise age of the Saint-Gerand-le-Puy and
Hessler viperids have been ambiguously associated to deposits that may come from either
MN1 or MN 2 [85?87], Weisenau vipers are still associated with MN 1 deposits . Thus,
the minimum age was constrained to 22.1 Mya (MN 1) based on the Weisenau viperid
vertebra. Maximum age was constrained to 93.9 Mya.
5. Crotalinae stem clade?Crotalinae gen. & sp. indet. A  was set as the MRCA of
Azemiops feae and Tropidolaemus wagleri. A left maxilla with an almost complete tooth
preserved in position (Department of Paleozoology, Institute of Zoology, Kiev, Ukraine, IZAN
3748) recovered from karstic fillings within a limestone quarry near the village of Gritsev,
Shepetovski district, Ukraine. The maxilla was assigned unambiguously to the Crotalinae
due to the deep depression on its posterolateral surface for the accomodation of the
thermoreceptive (pit) organ. Holman  and Holman and Tanimoto  reported possible
crown Crotalines from the lower Miocene of the U.S.A. and Japan, respectively. However,
despite the fact that there are no known records of viperines in Japan or the New World,
the identity of these older records as either crown or stem crotalines cannot be
unambiguously determined based on vertebral morphology alone. Thus, we retained the maxilla
described by Ivanov  as the only unambiguous crotaline record.
Ivanov  reported that the stratigraphic age of the site from Gritsev corresponds to the
middle Sarmatian MN 9a of Western Europe, with a minimum age of 10.4 Mya [
However, Vangengeim and Tesakov [
] argued that Gritsev was more accurately placed
within the upper part of the middle Sarmatian, which lies in a zone of reversed polarity that
is correlated to chron C5r. Therefore, we used the minimum age of 11.2 Mya estimated for
the boundary between the upper and middle Sarmatian and correlated with subchron
C5r.1n. The maximum age was constrained to 54 Mya.
6. Elapidae stem clade?Elapid Morphotype A [
] was set as the MRCA of Naja naja and
Buhoma depressiceps. One mostly complete posterior trunk vertebra (Tanzanian Antiquities
Unit, RRBP 04320) from locality TZ-01, Rukwa Rift Basin, southwestern Tanzania. This
specimen was referred to the family Elapidae due to its low and robust hypapophysis and
absence of a postzygapophyseal foramen [
]. It also shares a hypapophysis with a flattened
and laterally expanded ventral edge with some members of the genus Naja [
to McCartney et al. [
], the snake-bearing sites come from fluvial facies that belong to the
12 / 82
Songwe Member of the Nsungwe Formation, which is temporally constrained to ~ 24.9
Mya by mammalian biostratigraphy [
], detrital zircon geochronology, and a
radiometrically dated volcanic ashes [100?102]. Thus, we constrained the minimum age to 24.9
Mya. Maximum age is constrained to 54 Mya.
13 / 82
N. merkuriensis, N. sansaniensis) [117,118]. However, all of these taxa consisted of either
vertebral material or a few inconclusive cranial elements (e.g., compound bone, and
quadrate), and none retain well-preserved parabasisphenoids. Therefore, Natrix aff.
longivertebrata is here considered an unquestionable natricid record and is placed within colubroids,
as a crown Natricidae. The minimum age was constrained to 13.8 Mya and maximum age
to 54 Mya.
We compare our molecular data and phylogenetic results with three recently published
largescale molecular studies: Pyron et al. [
], Zheng and Wiens [
], and Figueroa et al. [
Pyron et al.?s [
] and Zheng and Wiens? [
] included representatives of all recognised
squamatan families, whereas Figueroa et al. [
] focused on snake lineages. Caenophidian coverage
in Pyron et al. [
] and Zheng and Wiens [
] were identical (1062 species) and included
sequences from up to 12 and up to 52 genes, respectively. Figueroa et al. [
] combined up to
10 genes for 1358 species of caenophidian snakes (excluding multiple individuals, unidentified,
and misidentified species). Our study combines sequences from up to 15 genes for 1263
species (see S3 Table for number of genes and accession numbers; see also S4 and S5 Tables for
more details on taxon sampling).
Higher-level relationships in these four large-scale studies are illustrated in Figs 1?3 and
discussed below. The comparisons among support metrics are discussed below and illustrated
in Figs 4 and 5. We also describe and illustrate separately the tree topology we obtained for
each well-supported colubroidean family (Figs 6?21). The full tree (including outgroups) is
provided as supporting information (S1 and S2 Figs). FBP and SHL, respectively, are provided
in parenthesis for each recovered clade discussed below and in Figs 6?21. When applicable,
the percentage of valid species sampled for a given genus is also shown in parentheses after
the name of the genus (see S4 and S5 Tables for a summary and a list of sampled species per
14 / 82
Fig 4. Scatterplots comparing support metrics for internal branches in the Maximum likelihood species-level phylogeny of Colubroides.
A) TBE and FBP B) SHL and FBP C) SHL and TBE, D) Histogram showing the proportion of each category of joint support in each comparison
of support metrics, E) Categories of joint support.
Comparison of support metrics
Support scores are strongly but imperfectly correlated. Pairwise scatterplots (Fig 4) show that
TBE and SHL scores tend to be higher (less conservative) than FBPs (see also S9 Table). Thus,
combining FBP and SHL produces almost the same proportion of supported clades as does
combining FBP and TBE, whereas combining TBE and SHL increases the number of
seemingly well-supported clades. This suggests that SHL and TBE tend to inflate and/or FBP tends
to underestimate the support for clades. Categories of combined support values can help
express such discrepancies by classifying all ambiguous supports as weakly supported clades
(gray points and bars in Fig 4). The tendency for SHL and TBE values to be higher than FBPs
affects all clade ages (Fig 5; S3 Fig). FBP tends to have weaker support values especially for
deeper nodes in our ML tree (S1 and S2 Figs).
15 / 82
Fig 5. Distribution of branch support scores for each node age based on the Maximum likelihood species-level phylogeny of Colubroides.
A) FBP distribution, B) SHL distribution, C) TBE distribution. Red dots represent values greater than 70%; gray dots indicate values smaller
16 / 82
Fig 6. Maximum likelihood species-level phylogeny of Colubroides. Families Xenodermidae, Pareidae, subfamily
Viperinae. Skeleton of the complete tree is displayed on the left, with the area of the tree corresponding to the present figure
highlighted in black. Colored squares on each node represent bootstrap and SHL values following the categories of combined
clade support described in S2 Table and summarized on the upper left corner of the figure. Diamonds on each tip represent
the percentage of data generated in this study for each terminal: white, 0%; light grey, between 1% and 50%; dark grey,
between 50% and 99%; black, 100%.
17 / 82
Fig 7. Maximum likelihood species-level phylogeny of Colubroides (continued). Family Viperidae, subfamilies Viperinae,
18 / 82
Fig 8. Maximum likelihood species-level phylogeny of Colubroides (continued). Family Viperidae, subfamily Crotalinae.
PLOS ONE | https://doi.org/10.1371/journal.pone.0216148 May 10, 2019 19 / 82
Fig 9. Maximum likelihood species-level phylogeny of Colubroides (continued). Family Viperidae, subfamily Crotalinae.
PLOS ONE | https://doi.org/10.1371/journal.pone.0216148 May 10, 2019 20 / 82
position is not statistically supported in our molecular tree, and it might be equally possible
for this species to cluster with Calamariidae since none of the branches that separate them
received significant combined SHL/BH support values (Fig 19). Therefore, in face of the
hemipenial and osteological similarities shared between Oreocalamus, Calamaria, and
Macrocalamus, we assign it to the Calamariidae instead of the Colubridae.
Colubroelaps?The monotypic genus Colubroelaps was described by Orlov et al.  to
accomodate a small fossorial snake from southern Vietnam. They provisionally included the
new genus in the family Colubridae (their subfamily Colubrinae) based mainly on the absence
of hypapophyses on the posterior trunk vertebrae and of a diastema and sulcate teeth on the
posterior end of the maxillae. However, the skull morphology of the type specimen shows that
C. nguyenvansangi has hinged teeth like sibynophiids (Fig W in S1 Appendix). Among
colubroidean snakes, Liophidium and Iguanognathus also retain hinged teeth [
], but differently
from the latter two genera, Colubroelaps shares with sibynophiids the presence of a distally
broadened, plate-like maxillary process of the palatine, absence of a choanal process of the
palatine, a long tubular dorsally-curved compound bone, reduced mandibular fossa, vestigial
splenial and angular bones, and a posterior dentigerous process of the dentary separated from
the compound bone and forming a projected free ending process that diverges from the main
mandibular axis (Figs V and W in S1 Appendix). Also, like Sibynophis and Scaphiodontophis,
the maxilla of Colubroelaps projects freely posteriorly to the maxillary-ectopterygoid contact,
but without forming an elongated ?dentigerous process? [
]. The combination of these
derived characters shared by Sibynophis, Scaphiodontophis, and Colubroelaps supports the
inclusion of the latter genus in the family Sibynophiidae. The absence of hypapophyses on the
posterior trunk vertebrae of C. nguyenvansangi seems to contradict the present allocation
because sibynophiids retain well-developed hypapophyses throuhought the trunk vertebrae
(Fig G in S2 Appendix). However, we suspect that posterior hypapophyses are reduced due to
the fossorial habits of that species.
Iguanognathus?The genus Iguanognathus was tentatively allocated in the families
Colubridae and Natricidae in the past, despite the lack of any compelling evidence supporting
either hypotheses. Like sibynophiids, Iguanognathus has hinged teeth , which would
suggest a possible close relationship with that family. However, apart from the presence of hinged
teeth, Iguanognathus does not share any of the other cranial specializations typical of
], as discussed above. Instead, Iguanognathus retains an aligned posterior
dentigerous process of the dentary, well developed, functional splenial and angular bones, a choanal
process, and a posteriorly curved tapering maxillary process of the palatine. Additionally,
unlike sibynophiids, Iguanognathus lacks hypapophyses on the posterior precloacal vertebrae
 which also rules out its belonging in the Natricidae. For these reasons, we prefer to
consider this genus a Colubridae incertae sedis.
A time-calibrated tree and the fossil record of Colubroides
Previous age estimates of Colubroides. Divergence time estimates have been increasingly
discussed in molecular studies of snake evolution in recent years [
Although they have been used to detail the tempo and mode of evolution of the group, these
studies have sometimes inferred substantially different dates for major events in colubroid
diversification. As Fig 23 shows, nine recent studies provide disparate dates for the origin of
most higher-level clades of colubroideans [22,58,124, 132?135,166]. Our divergence time
estimates are mostly concordant with those of Burbrink and Pyron  but are significantly
younger than the dates estimated by some other studies [
]. Although there are differences
in which fossils were used for calibrations, and in the numbers of genes and taxa, the different
61 / 82
results in Fig 23 mostly reflect expected differences between analyses based on autocorrelated
and uncorrelated molecular clocks . Studies in which time estimations were generated
by penalized likelihood algorithms (e.g. treePL) tend to keep the same general pattern and
same temporal cladogenic order. Comparing only the studies using autocorrelated methods
] and our own (Fig 23), we observe that a difference among time estimations for one
specific clade implies differences in the entire cladogenic process, that can be younger or older
as a whole, but can never be younger for some clades and at the same time older for others, or
vice-versa. In contrast, time estimations generated by uncorrelated methods [58,124,133,134]
and a Bayesian autocorrelation method  tend to be more variable with respect to the
general pattern, presenting cladogenic events that are ordered different among each study. As an
example, a previous analysis of the family Viperidae using an uncorrelated method  in
BEAST resulted in much older cladogenic events for the family as a whole (Fig 23). These
differences likely result from the different methods of divergence time estimation used by Alencar
et al.  and the present study.
Such disparate results suggest that inferred dates of divergence should be treated with
caution, and that the available fossil evidence is paramount to an accurate description of the
evolutionary trends of a group. Therefore, we integrate our estimated divergence dates with the
fossil record in an attempt to reach more balanced conclusions regarding the evolutionary
events underlying the origin and diversification of extant colubroidean families.
Despite their differences, some general trends emerge from the eight studies illustrated in
Fig 23. Nine of the ten studies estimated an early divergence time for the ancestor of
Colubroideans (i.e., the split between Colubroidea and Elapoidea) which, together, span an interval of
approximately 35 My, from the upper Cretaceous (Turonian) to the upper Paleocene
(Thanetian). Six of these studies place the origin of the group within the Cretaceous while three
retrieve a Paleocene origin. The former hypothesis of a Cretaceous origin of the group is
concordant with the presence of alleged colubroidean vertebral remains in the Cenomanian of the
Wadi Milk Formation of Sudan . However, the more complete material described by
Rage and Werner  belongs to the enigmatic Caenophidian family Russellophiidae, a
group known only from vertebral remains and only tentatively assigned to the clade
Colubroides. The other colubroidean vertebrae were considered of indeterminate Colubroidean
affinities due to their fragmentary condition , lacking preserved parts with unambiguous
derived colubroidean traits and rendering their assignment to this group questionable .
Although a late Cretaceous origin of the group seems likely, more definitive evidence of
Cretaceous colubroidean records is lacking.
Colubroidean early divergence. The colubroidean fossil record is mostly composed of
disarticulated vertebrae that are difficult to assign to any extant family because vertebral
characters alone are of limited value when it comes to diagnosing most colubroidean clades
]. Despite this limitation, colubroidean precloacal vertebrae were recognized until
recently by the following suite of derived characters, known to occur in combination only in
colubroidean snakes [
]: an elongate centrum, well-developed prezygapophyseal
accessory processes, a blade-like neural spine that extends anteriorly onto the zygosphene and
remains uniformly thin anteroposteriorly (as opposed to an expanded posterior margin of the
neural spine), well-developed subcotylar tubercles (or cotylar ventrolateral processes), distinct
dia- and parapophyseal articular facets of the synapophysis, prominent hypapophyses on
middle and posterior trunk vertebrae, and paracotylar foramina.
Among these characters, the uniformly thin blade-like neural spine that extends onto the
roof of the zygosphene appears to be invariably present in all colubroideans, and unique to the
group. However, our observations reveal that extant xenodermids lack an uniformly blade-like
neural spine that reaches the roof ot the zygosphene (Fig 25; Fig A in S2 Appendix) [
62 / 82
Although Achalinus, Xenodermus, and Fimbrios tend to retain a blade-like posterior margin,
the neural spine never invades the roof of the zygosphene anteriorly [
] (Fig 25; Fig A in
S2 Appendix). Therefore, we consider this character to represent a putative synapomorphy of
the clade Colubriformes, with important implications in the definition of the minimum age
used as calibration point for the base of our estimated colubroidean divergence time tree.
Among extinct putative caenophidian families Russellophiidae, Nigerophiidae, and
Anomalophiidae, only the latter seems to retain a similar neural spine morphology [
], and might
well represent an early colubriform lineage. However, the enigmatic nature of these three
families, known only from sparce vertebral material, precludes any unambiguous allocation to the
Therefore, the earliest records of allegedly uncontested colubroidean vertebrae from the
Lower to Upper Eocene [68,109,169?175] are more accurately assigned to the clade
Our time calibrated tree places the divergence of stem-colubroideans at ~ 56 Mya, near the
Paleocene/Eocene boundary, while stem-colubriforms diverged at ~ 53 Mya within the
Ypresian (Fig 22). Early fossil records of definitive colubriforms are concordant with this date,
with the oldest unequivocal record being of Procerophis sahnii  from the early Ypresian of
India, with an age of 54 Mya. (Fig 22) [
]. The other known Eocene colubriform
records are all from the middle/upper Eocene: an unnamed colubriform from the middle Eocene
of Namibia (41.2 Mya) ; an unnamed colubriform from the middle Eocene of Myanmar
(37.2 Mya) ; Renenutet enmerwer from the middle Eocene of Egypt (37 Mya) ; a
vertebra referred to Nebraskophis from the upper Eocene of Hardie Mine, USA (34.2 Mya) ;
and an unnamed colubriform from the upper Eocene of Thailand (34 Mya) . Because the
vertebrae of Vectophis wardi and Headonophis harrisoni from the upper Eocene of the Isle of
Wight, England (33.9 Mya) [170,173] retain a robust and posteriorly expanded neural spine
that does not invade the zygosphenal roof anteriorly, we treated them as caenophidians of
uncertain affinities instead of belonging to the clades Colubroides or Colubriformes.
Molecular evidence supports an early Paleogene divergence of colubroideans in Asia
[132,134], but they may have been present already in Africa in the early Upper Cretaceous
. The presence of a definitive colubriform snake in the Lower Eocene of Namibia ,
and the recent finding of Renenutet enmerwer in the Upper Eocene of Egypt  along with
an already well established colubroidean fauna in the Lower Oligocene of Tanzania [
indicates that the group had already diversified in Africa by the Eocene. Additionally, the presence
of diversilly significant number of colubriform records in India during the Eocene, including
the oldest undisputed colubriform snake, along with the African records discussed above,
suggest that colubroideans may have diversified much earlier in Gondwana prior to its dispersal
throughout Laurasia . In that context, the controversial presence of colubroidean snakes
in the Cenomanian of Sudan [
], which extends the divergence timing of the group to the
early Upper Cretaceous, seems to become a plausible hypothesis . However, additional
findings are needed to fill the implied ghost lineage of approximately 40 million years, from
the Upper Cretaceous sediments of the Wadi Milk Formation of Sudan to the Lower Eocene
undisputed colubriform record of India.
The Eocene-Oligocene transition and the diversification of present-day
Colubroid and Elapoid lineages
The early Oligocene was marked by a much cooler and more temperate global climate than the
warm "greenhouse" conditions that characterized most of the Cretaceous and early Cenozoic
[176,177]. The impoverished global diversity in the Oligocene that resulted from the
Eocene63 / 82
Oligocene extinctions is also observed in the fossil record of snakes around the world [
Our time calibrated tree for colubroideans illustrates this trend, with a relatively low number
of cladogenetic events dated prior to the Oligocene-Miocene boundary (Fig 22; S4 Fig).
However, these Oligocene cladogenetic events were key for the establishment of the present-day
colubroidean snake fauna. While an early divergence of basal colubroidean lineages is
estimated to have occurred within the Eocene, all extant families of Colubroidea and Elapoidea
that compose most of the present-day endoglyptodont fauna are estimated to have originated
rapidly within the early Oligocene interval, between ~ 33 and 28 Mya. (Figs 22 and 23; S4 Fig).
Diversification dates retrieved here are consistent with the major terrestrial faunal turnover
recorded around the world and associated with the overall climate shift at the
Eocene-Oligocene boundary. This trend is consistent with the sudden appearance in the European fossil
record of the derived colubrid vertebral morphotype with an elongated centrum, long
prezygapophyseal accessory processes, distinct epizygapophyseal spines, and a uniformly narrow
haemal keel (lacking hypapophyses), as illustrated by Coluber cadurci [
subsequent, mainly Miocene diversification of extant Colubroidean families, is also highly
consistent with the known Neogene colubroidean fossil record [32,178,179].
Among ?basal? colubroidean lineages estimated to have diverged within the Eocene (Fig
22), the Xenodermidae, Pareidae, and Homalopsidae still lack a fossil record, while the first
unequivocal viperid record is only early Miocene of age (MN1) , contrasting significantly
with our estimated timescale. Among Paleogene fossil caenophidian snakes, Thaumastophis
missiaeni approaches the xenodermid vertebral morphology in having vertically oriented
blade-like prezygapophyseal accessory processes and a lightly built and elongate vertebral
morphology . However, the combination of these two characters, along with the absence of
well-developed hypapophyses (present in xenodermids), and the presence of parazygantral
foramina (shared with acrochordids) and a blade-like neural spine invading the zygosphenal
tectum (shared with colubriformes) precludes its assignment to any of the three colubroidean
families cited above, or to the acrochordids . The lack of a well-established fossil record for
the Xenodermidae, Pareidae, and Homalopsidae during that interval of early colubroidean
evolution hampers any attempt to determine in more details the pattern of early divergence of
the group. Notwithstanding, according to our divergence estimates, it can be hypothesized
that appearance of grooved venomous teeth and the consequent diversification of higher
endoglyptodont lineages occurred within the Eocene, prior to the large-scale faunal turnover
that characterizes the Eocene-Oligocene transition [176,177,180?183]. Indeed, our time
calibrated tree indicates that stem-Xenodermidae diverged at ~ 52.6 Mya while stem-Pareidae and
stem-Endoglyptodonta diverged at ~ 45 Mya. Stem-Viperidae and stem-Homalopsidae also
diverged within the Eocene, at ~ 42.5 Mya and 38 Mya, respectively.
Viperidae fossil record and divergence time estimates. Although viperids are abundant
in the fossil record, most are confined to the Neogene, consist of isolated vertebrae, and are
assigned to extant taxa [79,89]. The oldest record of a viperid known so far is Provipera
boettgeri, described by Kinkelin  based on an isolated fang from the early Miocene of Germany
(MN1; ~ 21 to 23 Mya) (see Rage [
] for the validity of the name). Viperids are also recorded
in the early Miocene of Southern Asia (equivalent to MN 3)  and North America (late
Arikareean) , early or middle Miocene of Central Asia , and middle Miocene of
northern Africa (equivalent to MN 7+8) . The first unquestionable crotaline was reported
by Ivanov  from the middle Miocene of Gritsev in Ukraine (MN 9). Szyndlar and Rage
[79,85] provided a detailed review of the known Neogene fossil record of the family. As shown
by these authors, the fossil record of viperids does not help clarify the early divergence of the
family, since most fossils are associated with extant taxa from derived lineages [79,85], as
shown in our own phylogenetic tree (Figs 6?9).
64 / 82
Our time calibrated tree suggests that the origin of crown-Viperidae occurred in the early
Oligocene, at ~ 30.7 Mya, while the basal split between viperine and causine subfamilies on
the one hand, and crotaline and azemiopine subfamilies, on the other hand, occurred within
the late Oligocene, at approximately 26 and 25 Mya, respectively (Fig 22). As such, although
expected, no fossil viperids have been recorded yet in the Paleogene, resulting in an interval of
approximately 20 Mya between their hypothesized early divergence in the Eocene and the first
known, early Miocene, unequivocal fossil of the family [79,85]. Additionally, since the
sistergroup relationship between Asiatic/American crotalines and African/Eurasiatic viperines is
mainly symmetric, no conclusion can be reached on the geographic area of origin of the family
(apart from excluding the New World).
Elapoid fossil record and divergence time estimates. Within Elapoidea, only the
Elapidae and, possibly, Pseudoxyrhophiidae have a fossil record [
]. The oldest known
record of an unequivocal elapid comes from the late Oligocene of the Nsungwe Formation,
Tanzania, which bears sediments of ~ 25 Mya [
]. McCartney et al. [
] also report a distinct
caudal vertebra from the same locality in Tanzania that bears the unusual feature of a single
hemal keel instead of paired hemapophyses. According to the authors, the extant genus
Duberria also exhibits a similar condition, being the only known pseudoxyrhophiid snake so far that
lacks hemapophyses but retains a well-developed hemal keel. Although Duberria?s caudal
morphology ressembles the caudal vertebra described by McCartney et al. [
] as "Colubroid
Morphotype C", the authors rightly refrain from assigning the latter to Pseudoxyrhophiidae.
The oldest record of an Australian elapid consists of a vertebra attributed to a hydrophiine
found in sediments of Riversleigh dated from the upper Oligocene or lower Miocene (24?23
Mya) . According to Scanlon et al.  the vertebra is morphologically very similar to
the extant genus Laticauda. However, these authors refrain in allocating it to the latter genus
given the limited information afforded by one isolated vertebra.
The first record of elapids in Europe comes from the lower Miocene of France (MN 4;
]). Elapids are further abundantly documented throughout the Miocene and the Pliocene
of Europe [
], persisting in that continent until the upper Pliocene when they became
]. According to Szyndlar and Rage , most European fossil elapids are
assignable to extant Naja. This genus is also recorded in the middle Miocene of northern
Africa (13.8 Mya; MN70 [
]). The relatively abundant skull material found associated
with elapid vertebrae in the Neogene of Europe tend to corroborate the view that most of these
large Neogene elapids were either closely related to or nested within Naja [
Isolated posterior trunk vertebrae from the middle Miocene of North America (upper
Barstovian) and Europe (Astaracian, MN7) were assigned to extant Micrurus [
], as Micrurus
sp. and Micrurus gallicus, respectively. Referral to the family Elapidae is based on having a low
and recurved hypapophysis, a low anteroposteriorly elongated neural spine, and a poorly
vaulted neural arch. However, these characters may correlate well with fossorial habits [
and, thus, their assignment to the Micrurus is questionable. No compelling evidence
distinguishes the vertebral morphology of Micrurus from other Asian and Neotropical coral snake
genera. According to Rage and Holman [
], the few vertebrae referred to Micrurus from the
Miocene of Nebraska (USA) and la Grive (France) are comparable to extant Micrurus fulvius,
and thus should be referred to this genus. Our observations of the vertebral morphology of
South American Micrurus shows a very distinctive morphology from that of Micrurus fulvius,
suggesting that the vertebral morphology of the genus is much more diverse than previously
thought. A detailed description of the vertebral morphology of speciose New World Micrurus
and its closely related North American and Asiatic genera Micruroides, Leptomicrurus,
Calliophis, and Sinomicrurus is necessary to confidently support the assignment of these Miocene
records to any known extant genus, and especially to Micrurus.
65 / 82
Sub-Saharan elapids from the end of the Paleogene raise doubts about the well-accepted
hypothesis of an Asian origin for the group [
]. According to McCartney et al. , the
presence of elapids in the Nsungwe formation indicates two possible scenarios: a rapid initial
phase of dispersion of the family from Asia to Africa before the end of the Oligocene or,
alternatively, an origin of the family in Africa rather than Asia. The elapids from Nsungwe and
the hydrophiine from Riversleigh help reduce the gap between the fossil record and the most
recent molecular estimates (Fig 23). The presence of a hydrophiine in the late Oligocene or
early Miocene of Australia further supports the hypothesis of a dispersal and colonization of
the Australian continent in the late Oligocene [
Our time calibrated tree suggests an early divergence of stem-elapids within the early
Oligocene at ~ 30.5 Mya, with the main crown-Elapidae lineages diversifying during the late
Oligocene at ~ 26.5 Mya (Fig 22; S4 Fig). Although estimates of the time of divergence of Elapidae
seem to favour an early Oligocene origin (Fig 22), available molecular phylogenies (including
this one) and the fossil record do not yet allow inference of the biogeographic origin of the
group. While the discovery of elapids in the upper Oligocene of sub-Saharan Africa and the
unambiguous position of the family within the African elapoid radiation favour an African
origin, the basal-most positions of successive Asian coral-snake lineages in recent molecular
phylogenies tends to favor the opposite hypothesis of an Asian origin of the group. The lack of
support for deeper elapid relationship fails to provide a robust support for either hypothesis
Apart from the uncertainties involving the debate on an Asian or African origin, it has been
commonly thought that the family dispersed to the West Paleartic, Australian (via Melanesia),
and Neartic/Neotropical (via the Bering strait) regions independently [
Scanlon et al.  provided robust paleontological evidence supporting the hypothesis of an
over-water dispersal to Australia of the ancestor of the hydrophiine radiation, close to the
Oligocene?Miocene boundary. The dispersal into the West Paleartic in the lower Miocene of
forms belonging to or closely related to the extant genus Naja is also well documented
]. In contrast, the occurrence of extant genus Micrurus in the Miocene of
France is questionable due to the absence of vertebral diagnostic features that distinguish
members of this genus from the Asiatic coral snake radiation. Its record in the Miocene of
North America also needs further corroboration since Holman [
] used only two extant
species of Micrurus (M. fulvius and M. affinis) and Micruroides euryxanthus for comparison.
Our time calibrated tree places the early divergence of stem-hydrophiines at ~ 23 Mya, at
the Oligocene?Miocene boundary, and the ancestor of the New World radiation of coral
snakes (stem-micrurines) diverged from the Old World coral snakes at ~ 21 Mya in the early
Miocene. These results support the hypotheses of a late Oligocene over-sea dispersal and
colonization of the Australian continent by the ancestor of hydrophiines  and early Miocene
terrestrial colonization of North America by the ancestor of New World coral snakes.
Colubroid fossil record and divergence time estimates. The fossil record of Colubroidea
is much more extensive than that of elapoids but is mostly confined to the Neogene. Their
vertebral morphology remains poorly known, and an accurate evaluation of the fossils assigned to
this superfamily or to specific colubroid families remains far from being resolved.
Pseudoxenodontids, calamariids, sibynophiids, and grayiids have not been recorded so far in the fossil
record. In contrast, ?colubrid? and natricid fossils are abundant and have been recorded
throughout the Neogene of North America and Europe [
]. Colubrids and dipsadids
are also well-represented in the Neogene of North America .
Although Miocene and Pliocene records of colubroids are straightforward, Paleogene
records are more elusive and most of them are of uncertain assignment. Here we follow Smith
 in assigning the vertebrae from the upper Eocene of the Medicine Pole Hills of the
66 / 82
Chadron Formation in North Dakota to Colubroidea since they retain a ?racer-like? vertebral
morphology consistent with those of the North American racer clade of colubrids . This
record represents the oldest known Colubroidea so far.
The divergence between Colubroidea and Elapoidea in our time calibrated tree is estimated
at ~ 36 Mya, lying close to the boundary between the Eocene and Oligocene (Fig 22) and in
accordance with the appearance of colubroids in the late Eocene of North America
(Chadronian NALMA) . Similar to elapoid families in our time calibrated tree, ancestors of extant
families of colubroids diverged during the early Oligocene, between ~ 30 to 33 Mya. These
dates are also in agreement with the first emergence of the typical colubrid vertebral
morphotype in the early Oligocene of France, documented by Coluber cadurci from the Phosphorites
of Quercy (Mammal Paleogene Reference Unit MP 22). Colubrid precloacal vertebrae can be
distinguished from all other colubroidean family by the presence of the following combination
of derived features: an elongated centrum, long prezygapophyseal accessory processes, distinct
epizygapophyseal spines, and an uniformly narrow haemal keel (lacking hypapophyses).
Slightly younger records of putative natricids from the Phosphorites of Quercy are difficult to
allocate due to their fragmentary condition and their overall similarities with the vertebral
morphology of several extant elapoid families. Holman  and Szyndlar  detailed the
Neogene colubroid records in North America and Europe, respectively.
The traditional meaning of the superfamily Colubroidea [
] no longer
accommodates our growing knowledge of the phylogenetic affinities [
morphological disversity and disparities in the group [
]. Despite some pleas against
any change on the traditional usage of the names ?Colubroidea? and ?Colubridae? [
new morphological evidence provided here reinforces the need for a series of taxonomic
changes to accommodate new phylogenetic and morphological knowledge. Xenodermids,
pareids, and xylophiids represent ancient caenophidian lineages that are phylogenetically and
morphologically distinct from the endoglyptodont radiation. All three lineages lack the dental
specializations that gave rise to an advanced venom delivery system characteristic of
endoglyptodonts, thus breaking a universally accepted definition of colubroids as representing the truly
venomous snake radiation. Xenodermids further lack some vertebral and cranial features that
are commonly used to determine colubroid fossil remains and share with acrochordoids
cranial and vertebral specializations that are virtually absent in the remaining caenophidian
Our observations on caenophidian vertebral morphology, especially in xenodermids,
were particularly useful in redefining the fossil record commonly used as calibration points
in molecular phylogenies (e.g., the oldest colubroidean remains?Procerophis sahni?is here
placed as a calibration point for the early divergence between colubriformes and
xenodermids instead of the traditional divergence between xenodermids and acrochordids). By
reviewing and reinterpreting the relevant fossil record, our treePL analysis was able to
highlight previously unnoticed correlation between the early diversification of colubroid and
elapoid major lineages and the Eocene-Oligocene transition. Our divergence dates are in
general younger than most previous studies (Fig 23) and, although many authors would
suspect that disparate dates result from distinct methodological approaches, we suggest that
most of these differences are due primarily to the effect of highly heterogeneous usages of
the fossil record rather than of distinct methodological procedures. More importantly, our
results highlight the need for more detailed anatomical studies in combination with a more
careful usage of the fossil record [
67 / 82
Similarly, comparative morpho-functional or behavioural studies with caenophidians, that
are dependent on previously published, molecular phylogenies as frameworks, should seek
more accurate, combined evidence of branch support to evaluate their evolutionary scenarios.
Our statistical exploration of three different support methods indicates that TBE and SHL are
constantly higher and less conservative than FBP values. Based on the discrepancies among
these methods, we reinforce the combined use of different support values to identify nodes
that are not well supported or ambiguously supported. Such approach can help highlight
weakly supported clades and/or the presence of rogue terminals in phylogenetic datasets. Our
study revealed that a large number of colubroidean clades are still either poorly or
ambiguously supported and should be treated with caution.
S1 Table. Distinct classification schemes discussed in this study. Number of Phylogenetic
levels in each classification scheme are as follow: 1?3 = higher-levels, 4 = superfamily, 5 =
family, 6 = subfamily; families are listed in bold.
S2 Table. Categories of combined clade support. Graphic illustration for combined clade
support values when comparing FBP, SHL, and TBE metrics, classified in seven categories, as
follows: 1) Red, unambiguously supported; 2) Orange, robustly supported; 3) blue, strongly
supported; 4) green, moderately supported; 5) dark grey, ambiguously supported; 6) dark grey,
poorly supported; 7) light grey, unsupported (see text for discussion).
S3 Table. List of accession numbers. List of all the terminal taxa and sequences by gene used
in the present study, including best partitions, accession numbers of sequences retrieved from
GenBank and new sequences produced for this study.
S4 Table. Number and percentage of species and genera sequenced by family. Comparisons
between the number of families, genera and species of Colubroides used in the present study
and by Figueroa et al. [
S5 Table. Numbers and percentages for genera and species. Number of species by genus of
Colubroides sampled in this study; and a list of all species of Colubroides following Uetz et al.
], indicating which species was sampled in our study (spreadsheet 2).
S6 Table. List of sequences from GenBank considered questionable and/or problematical.
List of accession numbers, with genes names, current identification in GenBank, and probable
correct identification for questionable and/or problematic sequences of snakes available in
S7 Table. List of taxa recognized in this study but not listed by Uetz and Hosek (2017). List
with rationale for the species recognized in the present study, but not listed in Uetz et al. [
S8 Table. List of Primers used in this study. List with sequences for the pairs of primers used
to amplify the gene fragments used in the present study.
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S9 Table. Numbers of clades in each category of combined FBP, TBE, and SHL support
values. Clade support values based on the combination of (A) FBP/SHL, (B) FBP/TBE, (C) and
TBE/SHL, classified in seven categories, as follows: red, unambiguous support (both methods
recover values of 100%); orange, robust support (both methods recover values 90%,
or 80% in one method and 100% in the other); blue, strong support (both methods recover
values 80%, or values 70% in one method and 90% in the other); green, moderate
support (both methods recover values 70% but do not reach values equal to previous
categories); dark grey, ambiguous support (highly discrepant values, with < 70% in one method
and 80% in the other) or poor support (values < 70% in one method and between 70% and
80% in the other method); light grey, unsupported (values < 70% for both methods).
S10 Table. Representative fossil snakes from the Cenozoic. List of fossil snakes from the
Paleogene and Neogene with authorship, stratigraphic occurrence and locality.
S1 Fig. Full RAxML tree. Maximum likelihood tree of Colubroides containing 1263 terminals.
Color of the squares follow the categories of combined clade support as described in S2 Table.
Numbers inside de squares on the nodes of the full tree represent the bootstrap and SHL values
retrieved. Diamonds on each tip represent the percentage of the data for each terminal
generated in this study: white, 0%; light gray, between 1% and 50%; dark gray, between 50% and
99%; black, 100%. Terminals in red represent additional samples in relation to Pyron et al.
S2 Fig. Full treePL tree. Data matrix and calibrated tree resulting from the treePL analysis of
Colubroides, including the outgroups and containing 1278 terminals (1263 Colubroides and
S3 Fig. Full RAxML tree. Maximum likelihood species-level phylogeny of Colubroides
including comparisons among values of FBP, SHL, and TBE support metrics. Numbers inside de
squares on the nodes of the full tree represent the TBE values retrieved.
S4 Fig. treePL zoomed trees. Zoomed, large-scale calibrated tree resulting from the treePL
analysis showing the pattern of cladogenic events through time.
S1 Appendix. Skulls. Skull morphology of representatives of colubroidean families illustrating
the naso-frontal joint and optic foramen/fenestra. Figure A, Tropidophiidae: Tropidophis
nigriventris (AMNH 81182); Acrochordidae: Acrochordus granulatus (ZMB 9444). Figure B,
Xenodermidae: Achalinus spinalis (AMNH 34621), Fimbrios klossi (BMNH 19184.108.40.206).
Figure C, Xenodermidae: Xenodermus javanicus (FMNH 158613); Xylophiidae: Xylophis
perroteti (BMNH 19220.127.116.11). Figure D, Pareidae: Pareas moellendorffi (AMNH 27770),
Apopeltura boa (BMNH 47.12.30). Figure E, Viperidae: Azemiops kharini (ZMB 69985), Bothrops
neuwiedi (MZUSP 1476), Causus rhombeatus (FMNH 74241), Vipera ursinii (MZUSP 8230).
Figure F, Homalopsidae: Bitia hydroides (FMNH 229568), Brachyorrhos albus (FMNH
142322), Enhydris chinensis (AMNH 33870), Fordonia leucobalia (AMNH 107179). Figure G,
Homalopsidae: Homalopsis buccata (MNHN 1963.728); Psammophiidae: Malpolon
monspessulanus (AMNH 140768), Mimophis mahfalensis (UMMZ 209653). Figure H, Psammophiidae:
Psammophylax variabilis (AMNH 73213), Rhamphiophis oxyrhynchus (AMNH 16890),
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Psammophis phillipsi (AMNH 67750). Figure I, Cyclocoridae: Cyclocorus lineatus (MNHN
1900.413), Oxyrhabdium modestus (FMNH 53386); Atractaspididae: Aparallactus modestus
(AMNH 50545). Figure J, Atractaspididae: Atractaspis bibronii (AMNH 82073), Homoroselaps
lacteus (LSUMZ 57229), Macrelaps microlepidotus (FMNH 205860), Polemon christyi (FMNH
219913). Figure K, Lamprophiidae: Bothrolycus ater (AMNH 11976), Chamaelycus fasciatus
(BMNH 1909.4.29.3), Dipsadoboa weileri (AMNH 12472), Lamprophis olivaceus (AMNH
12003). Figure L, Lamprophiidae: Lycodonomorphus rufulus (AMNH 140284), Lycophidion
capense (AMNH 63771), Gonionotophis capensis (AMNH 73208), Pseudoboodon lemniscatus
(MNHN 1905.179). Figure M, Pseudoxyrhophiidae: Alluaudina bellyi (UMMZ 201605),
Dromicodryas quadrilineatus (UMMZ 209290), Duberria lutrix (UMMZ 154361), Heteroliodon
occipitalis (UMMZ 218178). Figure N, Pseudoxyrhophiidae: Ithycyphus miniatus (UMMZ
201615), Langaha madagascariensis (UMMZ 218193), Liophidium torquatum (UMMZ
209437), Pseudoxyrhopus tritaeniatus (UMMZ 203649). Figure O, Elapidae: Bungarus
caeruleus (AMNH 87483); Calliophis intestinalis (BMNH 1818.104.22.168), Micrurus narduccii
(MZUSP 8370), Naja naja (AMNH 86912). Figure P, Elapidae: Notechis scutatus (ZMB 7930),
Toxicocalamus loriae (AMNH 95581); Pseudoxenodontidae: Pseudoxenodon stricticaudatus
(AMNH 34674). Figure Q, Natricidae: Afronatrix anoscopa (MNHN 1986.1618), Aspidura
trachyprocta (AMNH 120251), Atretium schistosum (AMNH 85509), Lycognathophis seychellensis
(UMMZ 195836). Figure R, Natricidae: Natriciteres fuliginoides (MNHN 1987.1552), Natrix
maura (AMNH 115697), Sinonatrix annularis (AMNH 115693), Xenochrophis cerogaster
(AMNH 89276). Figure S, Dipsadidae: Apostolepis cf. nelsonjorgei (MZUSP 20636), Atractus
maculatus (IB 40003), Conophis pulcher (AMNH 117934), Contia tenuis (UMMZ 133519?1).
Figure T, Dipsadidae: Farancia abacura (KU 214419), Geophis hoffmanni (AMNH 113561),
Helicops pastazae (AMNH 49143), Heterodon nasicus (MNHN 1993.1625). Figure U,
Dipsadidae: Philodryas mattogrossensis (AMNH 141377), Sibon sartorii (LSUMZ 23243), Tachymenis
peruviana (KU 135193), Urotheca multilineata (AMNH 98284). Figure V, Dipsadidae:
Xenopholis scalaris (AMNH 60799); Sibynophiidae: Scaphiodontophis annulatus (MZUSP 5971),
Sibynophis subpunctatus (AMNH 96073). Figure W, Sibynophiidae: Colubroelaps
nguyenvansangi (ZISP/IEBR 25682). Figure X, Calamariidae: Calamaria gervaisi (AMNH 36744),
Macrocalamus lateralis (LSUMZ 45407); Oreocalamus hanitschi (BMNH 1922.214.171.124). Figure Y,
Grayiidae: Grayia smithii (AMNH 140428); Colubridae: Boiga dendrophila (AMNH 116014),
Coluber constrictor (FMNH 135284). Figure Z, Colubridae: Dendrelaphis papuensis (AMNH
107175), Ptyas mucosus (AMNH 83993), Scaphiophis albopunctatus (AMNH 104101),
Senticolis triaspis (AMNH 110625). Figure AA, Colubridae: Spilotes pullatus (IBSP 4955); Colubridae
incertae sedis: Iguanognathus werneri (BMNH 19126.96.36.199). Figure AB, Elapoidea incertae
sedis: Buhoma depressiceps (BMNH 1907.5.22.10), Micrelaps muelleri (HUJR 8009). Scale
bar = 1 mm.
S2 Appendix. Vertebrae. Posterior trunk vertebral morphology of representatives of
colubroidean families. Figure A, Acrochordidae: Acrochordus javanicus (USNM 297404), scale bar = 2
mm; Xenodermidae: Achalinus rufescens (BMNH 19188.8.131.52), scale bar = 1 mm; Fimbrios
klossi (BMNH 19184.108.40.206), scale bar = 1 mm; Pareidae: Pareas sp. (MZUSP 12186), scale
bar = 1 mm. Figure B, Viperidae: Causus difilippi (MZUSP 18668), scale bar = 5 mm; Vipera
ursinii (MZUSP 8230), scale bar = 5 mm; Azemiops feae (ROM 36976), scale bar = 1mm;
Bothrops jararaca (MZUSP 14425), scale bar = 2mm. Figure C, Homalopsidae: Cerberus
rynchops (MZUSP 9569), scale bar = 2mm; Homalopsis buccata (MZUSP 11483), scale bar = 1mm.
Psammophiidae: Psammophis lineolatus (MZUSP 8221), scale bar = 1mm; Mimophis
mahfalensis (MZSUP 12188), scale bar = 2mm. Figure D, Pseudoxyrhophiidae: Madagascarophis
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colubrinus (BMNH 220.127.116.11), scale bar = 2mm; Ditypophis vivax (BMNH_18.104.22.168), scale
bar = 1mm; Lamprophiidae: Boaedon fuliginosus (MZUSP 8167), scale bar = 2mm;
Crotaphopeltis hotamboeia (MZUSP 19602), scale bar = 1mm. Figure E, Atractaspididae: Atractaspis
irregulares (MZUSP 10826), scale bar = 1mm; Homoroselaps lacteus (LSUMZ 57229), scale
bar = 1mm. Elapidae: Sinomicrurus macclellandi (ROM 37113), scale bar = 1mm; Naja naja
(UMMZ 181137), scale bar = 1mm. Figure F, Elapidae: Micrurus corallinus (MZUSP 13112),
scale bar = 1mm; Cyclocoridae: Cyclocorus lineatus (BMNH 22.214.171.124), scale bar = 1mm;
Natricidae: Natrix natrix (MZUSP 2514), scale bar = 2mm; Natriciteres olivacea (MZUSP
2083), scale bar = 1mm. Figure G, Sibynophiidae: Scaphiodontophis annulatus (MZUSP 5971),
scale bar = 2mm; Grayiidae: Grayia smithii (MZUSP 8130), scale bar = 1mm; Grayia
tholloni (MZUSP 8135), scale bar = 2mm; Calamariidae: Oreocalamus hanitschi (BMNH
19126.96.36.199), scale bar = 1mm. Figure H, Colubridae: Chironius bicarinatus (MZUSP
13860), scale bar = 2mm; Spilotes pullatus (MZUSP 13845), scale bar = 2mm; Oxybelis aeneus
(MZUSP 13028), scale bar = 2mm; Mastigodryas boddaerti (MZUSP 13052), scale bar = 2mm.
Figure I, Colubridae: Simophis rhinostoma (MZUSP 13858), scale bar = 2mm; Dipsadidae:
Heterodon platirhinos (MZUSP 2991), scale bar = 2mm; Farancia abacura (MZUSP 2953),
scale bar = 2mm; Carphophis amoenus (MZUSP 8183), scale bar = 1mm. Figure J, Dipsadidae:
Synophis lasallei (MZUSP 7713), scale bar = 1mm; Nothopsis rugosus (MZUSP 7490), scale
bar = 1mm; Dipsas indica (IBSP 40137), scale bar = 1mm; Atractus serranus (MZUSP 17937),
scale bar = 1mm. Figure K, Dipsadidae: Boiruna maculata (MZUSP 703), scale bar = 2mm;
Helicops angulatus (MZUSP 14234), scale bar = 2mm; Philodryas nattereri (MZUSP 13039),
scale bar = 2mm; Oxyrhopus clathratus (MZUSP 14010), scale bar = 2mm.
S3 Appendix. Hemipenes. Hemipenial morphology of representatives of colubroidean
families. Figure A, Acrochordidae: Acrochordus javanicus (LSUMZ 34406) completely everted and
filled, scale bar = 5 mm; Xenodermidae: Xenodermus javanicus (FMNH 138678) partially
everted, partially filled, and dyed with alizarin red, scale bar = 1 mm. Figure B, Xenodermidae:
Achalinus rufescens (BMNH 1983.193) completely everted and partially filled, scale bar = 2
mm; Fimbrios klossi (BMNH 1965.2.639) opened through a longitudinal slit, one lobe
partially filled, scale bar = 1 mm; Pareidae: Pareas monticola (BMNH 1909.3.9.19) completely
everted and filled, scale bar = 1 mm. Figure C, Pareidae: Asthenodipsas malaccanus (BMNH
19188.8.131.52) completely everted and filled; Aplopeltura boa (BMNH 184.108.40.206) completely
everted and filled; scale bars = 2 mm. Figure D, Xylophiidae: Xylophis perroteti (BMNH
19220.127.116.11) opened through a longitudinal slit, spread flat, and dyed with alizarin red, scale
bar = 5 mm. Figure E, Viperidae: Porthidum nasutum (MZUSP 7480) completely everted and
filled; Vipera ammodytes (MZUSP 8223) completely everted and filled; scale bars = 5 mm.
Figure F, Viperidae: Bothrops neuwiedi (MZUSP 11851) completely everted and filled; Causus
bilineatus (MNHN 1993.5992) completely everted and filled; scale bars = 5 mm. Figure G,
Homalopsidae: Homalopsis buccata (MNHN 1963.728) completely everted and filled;
Brachyorrhos albus (FMNH 142324) completely everted and filled; scale bars = 5 mm. Figure H,
Homalopsidae: Fordonia leucobalia (AMNH 107179) completely everted, filled, and dyed with
alizarin red; Bitia hydroides (FMNH 229568) completely everted and filled; scale bars = 5 mm.
Figure I, Homalopsidae: Erpeton tentaculatum (AMNH 8850) completely everted and filled,
scale bar = 5 mm. Psammophiidae: Mimophis mahfalensis (UMMZ 209646) completely
everted and partially filled, scale bar = 2 mm; Atractaspididae: Polemon christyi (FMNH
219912) completely everted and filled, scale bar = 5 mm. Figure J, Atractaspididae: Atractaspis
fallax (AMNH 102298) completely everted and filled, scale bar = 10 mm; Macrelaps
microlepidotus (FMNH 205863) completely everted and filled, scale bar = 5 mm. Figure K,
71 / 82
Cyclocoridae: Cyclocorus lineatus (MNHN 1900.411) opened through a longitudinal slit,
spread flat, and dyed with alizarin red, scale bar = 5 mm; Oxyrhabdion modestum (FMNH
68907) completely everted, filled, and dyed with alizarin red, scale bar = 5 mm. Figure L,
Lamprophiidae: Lamprophis fuliginosus (MNHN 1994.8111) completely everted and filled, scale
bar = 5 mm; Chamaelycus fasciatum (BMNH 1909.4.29.2?3) completely everted and filled,
scale bar = 2 mm; Lycodonomorphus rufulus (AMNH 140283) completely everted and filled,
scale bar = 5 mm. Figure M, Lamprophiidae: Lycophidion semicinctus (MNHN 1995.3474)
completely everted, filled, and dyed with alizarin red; Mehelya capensis (AMNH 73208)
completely everted and filled; Pseudoboodon lemniscatus (MNHN 1905.185) completely
everted and filled; scale bars = 5 mm. Figure N, Pseudoxyrhophiidae: Dromicodryas bernieri
(UMMZ 218166) completely everted and filled, scale bar = 5 mm; Duberria lutrix (AMNH
115639) completely everted, filled, and dyed with alizarin red, scale bar = 3 mm; Alluaudina
bellyi (UMMZ 209239) completely everted and filled, scale bar = 2 mm. Figure O,
Pseudoxyrhophiidae: Pseudoxyrhopus tritaeniatus (UMMZ 195854) completely everted and filled;
Liophidium torquatum (UMMZ 209430) completely everted and filled; scale bars = 5 mm. Figure P,
Elapidae: Naja melanoleuca (BMNH 1918.104.22.168) completely everted and filled; Micrurus
frontalis (IBSP 44331) completely everted and filled; scale bars = 5 mm. Figure Q, Elapidae:
Austrelaps superbus (BMNH 1922.214.171.124) completely everted and filled; Bungarus candidus
(BMNH 1937.11) completely everted and filled; scale bars = 5 mm. Figure R, Natricidae:
Atretium schistosum (AMNH 85505) completely everted, filled, and dyed with alizarin red;
Lycognathophis seychellensis (UMMZ 167994) completely everted, filled, and dyed with alizarin red;
Afronatrix anoscopus (AMNH 142404) completely everted, filled, and dyed with alizarin red;
scale bars = 2 mm. Figure S, Natricidae: Xenochrophis vittatus (BMNH 126.96.36.199?6)
completely everted and filled; Natriciteres olivacea (AMNH 11905) completely everted and
filled; Sinonatrix annularis (AMNH 84530) completely everted, filled, and dyed with alizarin
red; scale bars = 2 mm. Figure T, Natricidae: Aspidura trachyprocta (AMNH 120248)
completely everted and partially filled (no scale); Elapoidis fusca (MNHN 1895.55) completely
everted and filled, scale bar = 2 mm. Figure U, Pseudoxenodontidae: Pseudoxenodon macrops
(AMNH 34649) completely everted and filled; Dipsadidae: Conophis pulcher (MNHN 5981)
completely everted and filled; scale bars = 5 mm. Figure V, Dipsadidae: Contia tenius (UMMZ
133370) completely everted and filled, scale bar = 2 mm; Urotheca decipiens (KU 103892)
completely everted and filled, scale bar = 5 mm. Figure W, Dipsadidae: Oxyrhopus occipitalis
(AMNH 129255) completely everted, filled, and dyed with alizarin red; Farancia
erythrogramma (KU 197245) completely everted, filled, and dyed with alizarin red. Scale bars = 5
mm. Figure X, Dipsadidae: Tachymenis chilensis (MZUSP 8239) completely everted, filled, and
dyed with alizarin red, scale bar = 2 mm; Heterodon nasicus (MNHN 3636) completely everted
and filled, scale bar = 5 mm; Philodryas olfersii (IBSP 63455) completely everted, filled, and
dyed with alizarin red, scale bar = 5 mm. Figure Y, Sibynophiidae: Sibynophis chinensis
(AMNH 34102) completely everted and filled, scale bar = 2 mm; Scaphiodontophis annulatus
(KU 191073) completely everted and filled, scale bar = 2 mm; Calamariidae: Pseudorabdion
longiceps (BMNH 1969.1866) completely everted and partially filled, scale bar = 5 mm.
Figure Z, Calamariidae: Calamaria lumbricoidis (BMNH 19188.8.131.52) completely everted and
partially filled, scale bar = 5 mm; Calamaria linnaei (AMNH 31943) completely everted and
partially filled, scale bar = 1 mm; Oreocalamus hanitschi (BMNH 19184.108.40.206) completely
everted and partially filled, scale bar = 5 mm. Figure AA, Grayiidae: Grayia ornata (BMNH
220.127.116.11) completely everted and filled; Colubridae: Pantherophis guttatus (USNM 523605)
completely everted and filled; Spilotes sulphureus (IBSP 68260) completely everted and filled.
Scale bars = 10 mm. Figure AB, Colubridae: Dispholidus typus (AMNH 23110) completely
everted and filled; Hierophis viridiflavus (MNHN 1978.414) completely everted and filled;
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Boiga pulverulenta (MNHN 1967.437) completely everted and filled; scale bars = 10 mm.
Figure AC, Colubridae: Ptyas korros (AMNH 84460) completely everted and filled, scale
bar = 10 mm; Gongylosoma baliodeirus (MNHN 1989.199) completely everted and filled,
scale bar = 3 mm; Liopeltis frenatus (MNHN 1928.75) completely everted and filled, scale
bar = 5 mm. Figure AD, Elapoidea Incertae sedis: Buhoma depressiceps (MNHN 1991.1740)
completely everted, partially filled, and dyed with alizarin red, scale bar = 1 mm.
The authors wish to thank the following colleagues who kindly supplied tissue samples and/or
allowed access to the specimens under their care: CW Myers, DR Frost, D Kizirian (AMNH);
K de Queiroz, R McDiarmid, G Zug (USNM); A Dubois, A Ohler, I Ineich, R Bour (MNHN);
P Campbell, D Gower (BMNH); MT Rodrigues (IBUSP); W Duellman, L Trueb (KU); D.
Rossman, J. Boundy (LSUMZ); R. MacCulloch, A. Lathrop (ROM); M-O Ro?del, F Tillack, R
Gu?nther (ZMB); A Resetar (FMNH); R Nussbaum, G Schneider (UMMZ); G Puorto, FL
Franco (IBSP); B Schacham, Y Werner (HUJR). We are especially indebted to AB Carvalho
and R Rodrigues for scanning specimens and providing technical support for figure
preparations, to L. Oliveira for helping with the scanning electron microscopy of maxillary teeth, and
to T. Rowe and J. Maisano for providing 3D reconstructions of squamate taxa scanned as part
of the DigiMorph project. We are grateful to C Sarturi and A Schnorr (PUCRS) and Dr. Y
Gao, P-T Luan and J-X Wang (KIZ) for laboratory assistance. FGG. and RG were supported
by scholarships from Fundac??o de Amparo ? Pesquisa do Estado de S?o Paulo (FAPESP grant
numbers 2007/52781-5, 2012/ 08661?3, 2007/52144-5, 2011/2167-4, 2008/52285-0, 2012/
24755-8, 2016/13469-5). Funding for this study was provided by FAPESP (2002/13602-4,
2011/50206-9 and 2016/50127-5).
Conceptualization: Hussam Zaher, Robert W. Murphy, Felipe G. Grazziotin.
Data curation: Hussam Zaher, Roberta Graboski, Kristin Mahlow, Felipe G. Grazziotin.
Formal analysis: Hussam Zaher, Roberta Graboski, Mark Wilkinson, Felipe G. Grazziotin.
Funding acquisition: Hussam Zaher.
Investigation: Hussam Zaher, Mark Wilkinson, Felipe G. Grazziotin.
Methodology: Hussam Zaher, Mark Wilkinson, Felipe G. Grazziotin.
Project administration: Hussam Zaher, Ya-Ping Zhang.
Resources: Hussam Zaher, Robert W. Murphy, Ya-Ping Zhang, Felipe G. Grazziotin.
Software: Felipe G. Grazziotin.
Supervision: Hussam Zaher, Robert W. Murphy, Ya-Ping Zhang, Felipe G. Grazziotin.
Validation: Robert W. Murphy, Juan Camilo Arredondo, Roberta Graboski, Paulo Roberto
Machado-Filho, Giovanna G. Montingelli, Ana Bottallo Quadros, Nikolai L. Orlov, Mark
Wilkinson, Ya-Ping Zhang, Felipe G. Grazziotin.
Visualization: Juan Camilo Arredondo, Roberta Graboski, Paulo Roberto Machado-Filho,
Kristin Mahlow, Giovanna G. Montingelli, Ana Bottallo Quadros, Nikolai L. Orlov, Mark
Wilkinson, Ya-Ping Zhang, Felipe G. Grazziotin.
73 / 82
Writing ? original draft: Hussam Zaher, Juan Camilo Arredondo, Roberta Graboski, Paulo
Roberto Machado-Filho, Giovanna G. Montingelli, Ana Bottallo Quadros, Mark
Wilkinson, Felipe G. Grazziotin.
Writing ? review & editing: Robert W. Murphy, Juan Camilo Arredondo, Roberta Graboski,
Paulo Roberto Machado-Filho, Kristin Mahlow, Giovanna G. Montingelli, Ana Bottallo
Quadros, Nikolai L. Orlov, Mark Wilkinson, Ya-Ping Zhang, Felipe G. Grazziotin.
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